A pyridoxal phosphate-dependent enzyme platform for stereoselective synthesis of γ-tertiary-nitro-noncanonical amino acids

Non-canonical amino acids (ncAAs) are privileged building blocks for the synthesis of natural products, biocatalysts, and drug molecules. Among them, γ-tertiary-nitro-α-amino acids are highly valuable yet remain scarcely explored due to challenges in synthetic accessibility. Although pyridoxal-5'-phosphate (PLP)-dependent enzymes are powerful biocatalysts for ncAA production, no enzymatic platform has, to the best of our knowledge, enabled efficient access to γ-tertiary-nitro-α-amino acids. Here, we report a PLP-dependent enzymatic platform that selectively couples O-acetyl-serine with secondary nitroalkanes to yield γ-tertiary-nitro-α-amino acids. Through reaction design, enzyme screening, and directed evolution, we repurpose SidM, an enzyme that catalyzes pyrrole ring formation in the siderochelin biosynthetic pathway, into a highly active and stereoselective biocatalyst, affording γ-tertiary-nitro-α-amino acids in up to 99% yield and 99% diastereomeric excess. This enzyme exhibits broad substrate scope and good evolvability, allowing enhancement of catalytic efficiency or inversion of diastereoselectivity at the Cγ nitro center, and is readily scalable to gram synthesis. Mechanistic analyses combining computation and mutagenesis elucidate the structural determinants of activity and stereocontrol. This work establishes a general, tunable and scalable biocatalytic platform for the synthesis of γ-tertiary-nitro-α-amino acids, expanding the synthetic repertoire of PLP-dependent enzymes for asymmetric C-C bond formation.